Biomarker of depression, method for measuring biomarker of depression, computer program, and recording medium

ABSTRACT

A method for treating depression using a biomarker includes measuring a level of phosphoethanolamine in a blood sample collected from a subject, comparing the level of phosphoethanolamine in the blood sample with a predetermined threshold, in response to the level of phosphoethanolamine in the blood sample being below the predetermined threshold, determining that the subject requires a treatment for depression, and treating the subject determined to suffer from depression.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of the priority to theJapanese Patent Applications No. 2009-187521 filed on Aug. 12, 2009 andNo. 2009-291029 filed on Dec. 22, 2009, disclosures of which are hereinincorporated by reference.

TECHNICAL FIELD

The present invention relates to biomarkers for diagnosing depression.

BACKGROUND ART

The depression is one type of mood disorders, and its predominantsymptoms are the “depressed mood” and “loss of interest or pleasure”,but some patients complain other symptoms such as suppressed appetite,insomnia, fatigue, suicidal ideation etc. Its diagnosis is generallyconducted based on the criterion under either ICD-10 (InternationalClassification of Diseases) by WHO (World Health Organization) or“DSM-IV” by American Psychiatric Association.

However, the diagnosis of depression can be hardly objective because itdepends on the impression by physician or psychologists, or impressionby the patients or individuals complaining the symptom or stress. Infact, cases are often observed where symptoms are excessively reportedeither intentionally or unintentionally because of the disease gain inwhich a patient is somehow benefitted from the state of being sick, orotherwise symptoms are concealed for the purpose of avoiding prejudiceor trouble from being known to be in depression. In such cases, accuratediagnosis of the disease is difficult, and determination of treatmentmethod may become difficult, or even inappropriate treatment might beapplied due to misdiagnosis.

Therefore, several methods have been tested to objectively assist thediagnosis by physician or psychologists. One example is the method inwhich the noise appearing in the symptom of a patient is excluded asmuch as possible by processing the results of diagnosis made by aphysician or psychologist with a computer (WO2004/080312).

Several markers for depression are known, including macromolecularmaterials which have been reported in the method to measure theexpression level of a gene (JP2008-253258) and the method to detectproteolytic products (JP2009-92550).

On the other hand, smaller compounds in living bodies are also tested ascandidates for the depression marker, including those having beenreported in the method to measure and analyze the contents oftestosterone and cortisol (JP2007-24822) and the method to detect thedegraded products of tryptophan in vivo (WO2006/105907), but none ofthem is brought into practice use so far.

SUMMARY OF INVENTION Technical Problem

The present invention is aimed to provide biomarkers for diagnosingdepression.

Solution to Problem

The present invention consists of the following Items.

-   (1) A biomarker for diagnosing depression, wherein the biomarker is    selected from the group consisting of ADP-RIBOSE, ATP, ADP, AMP,    serotonin, tryptophan, kynurenine, SDMA (symmetric    dimethylarginine), threonine, glyceric acid, serine,    N-acetylaspartic acid, glutamic acid, trigonelline, creatine,    2-methyl serine, sphingosine, homovanillic acid, piperidine,    sulfoxidated methionine, pipecolic acid, sphinganine,    γ-butyrobetaine, guanidoacetic acid, isobutyric acid, creatinine,    sarcosine, 3-methyl butyric acid, nicotinamide, betaine, ornithine,    carnitine, ethanolamine, phosphoethanolamine, taurine, hypotaurine,    aspartic acid, methionine, tyrosine, phosphorylcholine, arginine,    asparagine, 3-aminobutyric acid, β-alanine, phenylalanine, lysine,    phosphocreatine, alanine, uric acid, choline, isocitric acid,    leucine, malic acid, aminoadipic acid, tyramine, valine, glucaric    acid, histidine, ADMA (asymmetric dimethylarginine), isoleucine,    hydroxyproline and cystathionine.-   (2) The biomarker for diagnosing depression according to Item (1),    wherein the biomarker is selected from the group consisting of    phosphoethanolamine, taurine, hypotaurine, aspartic acid,    methionine, tyrosine, phosphorylcholine, arginine, asparagine,    3-aminobutyric acid, β-alanine, phenylalanine, lysine,    phosphocreatine, alanine, uric acid, choline, isocitric acid,    leucine, malic acid, aminoadipic acid, tyramine, valine, glucaric    acid, histidine, ADMA, isoleucine, hydroxyproline and cystathionine.-   (3) The biomarker for diagnosing depression according to Item (1),    wherein the biomarker is selected from the group consisting of    phosphoethanolamine, taurine, aspartic acid, methionine, tyrosine,    aminoadipic acid, histidine and isoleucine.-   (4) The biomarker for diagnosing depression according to Item (1),    wherein the biomarker is selected from the group consisting of    hypotaurine, phosphorylcholine, arginine, 3-aminobutyric acid,    β-alanine, lysine, phosphocreatine, alanine, uric acid, choline,    isocitric acid, leucine, malic acid, tyramine, valine and ADMA.-   (5) The biomarker for diagnosing depression according to Item (1),    wherein the biomarker is selected from the group consisting of    asparagine, phenylalanine, glucaric acid, hydroxyproline and    cystathionine.-   (6) The biomarker for diagnosing depression according to Item (1),    wherein the biomarker is cystathionine.-   (7) The biomarker for diagnosing depression according to Item (1),    wherein the biomarker is selected from the group consisting of ADMA,    hypotaurine, lysine, histidine, leucine, β-alanine, choline,    threonine, glyceric acid, isocitric acid, serine, malic acid,    2-methyl serine, sphingosine, homovanillic acid, sulfoxidated    methionine, sphinganine, phenylalanine, alanine, 2-aminoadipic acid,    N-acetylaspartic acid, glutamic acid, trigonelline, creatine,    guanidoacetic acid, isobutyric acid, creatinine, sarcosine, betaine,    phosphoethanolamine, taurine, ADP-RIBOSE, aspartic acid, tyrosine,    ATP, ADP, asparagine, AMP, serotonin, valine, tryptophan,    kynurenine, phosphorylcholine, isoleucine, SDMA, piperidine,    pipecolic acid, creatine, nicotinamide, γ-butyrobetaine, uric acid,    3-methyl butyric acid, 3-aminobutyric acid, ornithine, carnitine,    ethanolamine and methionine.-   (8) The biomarker for diagnosing depression according to Item (1),    wherein the biomarker is selected from the group consisting of    methionine, phosphoethanolamine, taurine, ADP-RIBOSE, aspartic acid,    tyrosine, ATP, ADP, asparagine, AMP, serotonin, valine, tryptophan,    kynurenine, phosphorylcholine, isoleucine, SDMA, piperidine,    pipecolic acid, creatinine and nicotinamide.-   (9) The biomarker for diagnosing depression according to Item (1),    wherein the biomarker is selected from the group consisting of ADMA,    hypotaurine, lysine, histidine, leucine, β-alanine, choline,    threonine, glyceric acid, isocitric acid, serine, malic acid,    2-methyl serine, sphingosine, homovanillic acid, sulfoxidated    methionine, sphinganine, isobutyric acid, γ-butyrobetaine, uric    acid, 3-methyl butyric acid, 3-aminobutyric acid, ornithine,    carnitine and ethanolamine.-   (10) The biomarker for diagnosing depression according to Item (1),    wherein the biomarker is selected from the group consisting of    phenylalanine, alanine, aminoadipic acid, N-acetylaspartic acid,    glutamic acid, trigonelline, creatine, guanidoacetic acid, sarcosine    and betaine.-   (11) A method of measurement including the step of measuring a    content of one or more compounds selected from the group consisting    of ADP-RIBOSE, ATP, ADP, AMP, serotonin, tryptophan, kynurenine,    SDMA (symmetric dimethylarginine), threonine, glyceric acid, serine,    N-acetylaspartic acid, glutamic acid, trigonelline, creatine,    2-methyl serine, sphingosine, homovanillic acid, piperidine,    sulfoxidated methionine, pipecolic acid, sphinganine,    γ-butyrobetaine, guanidoacetic acid, isobutyric acid, creatinine,    sarcosine, 3-methyl butyric acid, nicotinamide, betaine, ornithine,    carnitine, ethanolamine, phosphoethanolamine, taurine, hypotaurine,    aspartic acid, methionine, tyrosine, phosphorylcholine, arginine,    asparagine, 3-aminobutyric acid, β-alanine, phenylalanine, lysine,    phosphocreatine, alanine, uric acid, choline, isocitric acid,    leucine, malic acid, aminoadipic acid, tyramine, valine, glucaric    acid, histidine, ADMA (asymmetric dimethylarginine), isoleucine,    hydroxyproline and cystathionine in a collected blood sample.-   (12) The method of measurement according to Item (11), the method    including the step of measuring a content of one or more compounds    selected from the group consisting of phosphoethanolamine, taurine,    hypotaurine, aspartic acid, methionine, tyrosine, phosphorylcholine,    arginine, asparagine, 3-aminobutyric acid, β-alanine, phenylalanine,    lysine, phosphocreatine, alanine, uric acid, choline, isocitric    acid, leucine, malic acid, aminoadipic acid, tyramine, valine,    glucaric acid, histidine, ADMA, isoleucine, hydroxyproline and    cystathionine in a collected blood sample.-   (13) The method of measurement according to Item (11), the method    including the steps of:

measuring a content of a first compound selected from the groupconsisting of hypotaurine, aspartic acid, methionine, tyrosine,phosphorylcholine, arginine, asparagine, 3-aminobutyric acid, β-alanine,phenylalanine, lysine, phosphocreatine, alanine, uric acid, choline,isocitric acid, leucine, malic acid, aminoadipic acid, tyramine, valine,glucaric acid, histidine, ADMA, isoleucine, hydroxyproline andcystathionine in a collected blood sample, and measuring a content of asecond compound selected from the group consisting ofphosphoethanolamine and taurine in the blood sample.

-   (14) The method of measurement according to Item (11), the method    including the step of measuring a content of aspartic acid and    arginine in the blood sample.-   (15) The method of measurement according to Item (11), the method    including the step of measuring a content of aspartic acid and    tyrosine in the blood sample.-   (16) The method of measurement according to Item (11), the method    including the step of measuring a content of tyrosine and glucaric    acid in the blood sample.-   (17) The method of measurement according to Item (11), the method    including the step of measuring a content of tyrosine and    3-aminobutyric acid in the blood sample.-   (18) The method of measurement according to Item (11), the method    including the step of measuring a content of one or more compounds    selected from the group consisting of ADMA, hypotaurine, lysine,    histidine, leucine, β-alanine, choline, threonine, glyceric acid,    isocitric acid, serine, malic acid, 2-methyl serine, sphingosine,    homovanillic acid, sulfoxidated methionine, sphinganine,    phenylalanine, alanine, 2-aminoadipic acid, N-acetylaspartic acid,    glutamic acid, trigonelline, creatine, guanidoacetic acid,    isobutyric acid, creatinine, sarcosine, betaine,    phosphoethanolamine, taurine, ADP-RIBOSE, aspartic acid, tyrosine,    ATP, ADP, asparagine, AMP, serotonin, valine, tryptophan,    kynurenine, phosphorylcholine, isoleucine, SDMA, piperidine,    pipecolic acid, creatine, nicotinamide, γ-butyrobetaine, uric acid,    3-methyl butyric acid, 3-aminobutyric acid, ornithine, carnitine,    ethanolamine and methionine in a collected blood sample.-   (19) A method for judging an effect of a drug for depression, the    method including the steps of:

measuring a content of one or more compounds in blood samples collectedfrom a patient afflicted with the depression before and after anadministration of the drug to the patient, and

comparing the content of the compound between the blood samples beforeand after the administration,

wherein the compound is selected from the group consisting ofADP-RIBOSE, pairs of ATP, ADP, AMP, serotonin, tryptophan, kynurenine,SDMA (symmetric dimethylarginine), threonine, glyceric acid, serine,N-acetylaspartic acid, glutamic acid, trigonelline, creatine, 2-methylserine, sphingosine, homovanillic acid, piperidine, sulfoxidatedmethionine, pipecolic acid, sphinganine, γ-butyrobetaine, guanidoaceticacid, isobutyric acid, creatinine, sarcosine, 3-methyl butyric acid,nicotinamide, betaine, ornithine, carnitine, ethanolamine,phosphoethanolamine, taurine, hypotaurine, aspartic acid, methionine,tyrosine, phosphorylcholine, arginine, asparagine, 3-aminobutyric acid,β-alanine, phenylalanine, lysine, phosphocreatine, alanine, uric acid,choline, isocitric acid, leucine, malic acid, aminoadipic acid,tyramine, valine, glucaric acid, histidine, ADMA (asymmetricdimethylarginine), isoleucine, hydroxyproline and cystathionine.

-   (20) The method for judging an effect of a drug for depression    according to Item (19), the method including the steps of:

measuring a content of one or more compounds in blood samples collectedfrom a patient afflicted with the depression before and after anadministration of the drug to the patient, and

comparing the content of the compound between the blood samples beforeand after the administration,

wherein the compound is selected from the group consisting ofphosphoethanolamine, taurine, hypotaurine, aspartic acid, methionine,tyrosine, phosphorylcholine, arginine, asparagine, 3-aminobutyric acid,β-alanine, phenylalanine, lysine, phosphocreatine, alanine, uric acid,choline, isocitric acid, leucine, malic acid, aminoadipic acid,tyramine, valine, glucaric acid, histidine, ADMA, isoleucine,hydroxyproline and cystathionine.

-   (21) A method for judging an effect of a drug for a depression, the    method including the steps of:

measuring a content of one or more compounds in a blood sample drawnfrom a patient afflicted with the depression after an administration ofthe drug to the patient, and

comparing a threshold for a content of the compound in blood todistinguish a patient with depression from a healthy subject with themeasured content of the compound,

wherein the compound is selected from the group consisting ofADP-RIBOSE, ATP, ADP, AMP, serotonin, tryptophan, kynurenine, SDMA(symmetric dimethylarginine), threonine, glyceric acid, serine,N-acetylaspartic acid, glutamic acid, trigonelline, creatine, 2-methylserine, sphingosine, homovanillic acid, piperidine, sulfoxidatedmethionine, pipecolic acid, sphinganine, γ-butyrobetaine, guanidoaceticacid, isobutyric acid, creatinine, sarcosine, 3-methyl butyric acid,nicotinamide, betaine, ornithine, carnitine, ethanolamine,phosphoethanolamine, taurine, hypotaurine, aspartic acid, methionine,tyrosine, phosphorylcholine, arginine, asparagine, 3-aminobutyric acid,β-alanine, phenylalanine, lysine, phosphocreatine, alanine, uric acid,choline, isocitric acid, leucine, malic acid, aminoadipic acid,tyramine, valine, glucaric acid, histidine, ADMA (asymmetricdimethylarginine), isoleucine, hydroxyproline and cystathionine.

-   (22) The method for judging an effect of a drug for a depression    according to Item (21), the method including the steps of:

measuring a content of one or more compounds in a blood sample drawnfrom a patient afflicted with the depression after an administration ofthe drug to the patient, and

comparing a threshold for a content of the compound in blood todistinguish a patient with depression from a healthy subject with themeasured content of the compound,

wherein the compound is selected from the group consisting ofphosphoethanolamine, taurine, hypotaurine, aspartic acid, methionine,tyrosine, phosphorylcholine, arginine, asparagine, 3-aminobutyric acid,β-alanine, phenylalanine, lysine, phosphocreatine, alanine, uric acid,choline, isocitric acid, leucine, malic acid, aminoadipic acid,tyramine, valine, glucaric acid, histidine, ADMA, isoleucine,hydroxyproline and cystathionine.

-   (23) A method for determining a threshold for a content of one or    more compounds in blood to distinguish a patient with depression    from a healthy subject, the method including the step of measuring a    content of the compound in blood samples collected from plurality of    patients diagnosed as being afflicted with depression and plurality    of healthy subjects,

wherein the compound is selected from the group consisting ofADP-RIBOSE, ATP, ADP, AMP, serotonin, tryptophan, kynurenine, SDMA(symmetric dimethylarginine), threonine, glyceric acid, serine,N-acetylaspartic acid, glutamic acid, trigonelline, creatine, 2-methylserine, sphingosine, homovanillic acid, piperidine, sulfoxidatedmethionine, pipecolic acid, sphinganine, γ-butyrobetaine, guanidoaceticacid, isobutyric acid, creatinine, sarcosine, 3-methyl butyric acid,nicotinamide, betaine, ornithine, carnitine, ethanolamine,phosphoethanolamine, taurine, hypotaurine, aspartic acid, methionine,tyrosine, phosphorylcholine, arginine, asparagine, 3-aminobutyric acid,β-alanine, phenylalanine, lysine, phosphocreatine, alanine, uric acid,choline, isocitric acid, leucine, malic acid, aminoadipic acid,tyramine, valine, glucaric acid, histidine, ADMA (asymmetricdimethylarginine), isoleucine, hydroxyproline and cystathionine.

-   (24) The method for determining a threshold of a content of one or    more compounds in blood to distinguish a patient with depression    from a healthy subject according to Item (23), the method including    the step of:

measuring a content of the compound in blood samples collected fromplurality of patients diagnosed as being afflicted with depression andplurality of healthy subjects,

wherein the compound is selected from the group consisting ofphosphoethanolamine, taurine, hypotaurine, aspartic acid, methionine,tyrosine, phosphorylcholine, arginine, asparagine, 3-aminobutyric acid,β-alanine, phenylalanine, lysine, phosphocreatine, alanine, uric acid,choline, isocitric acid, leucine, malic acid, aminoadipic acid,tyramine, valine, glucaric acid, histidine, ADMA, isoleucine,hydroxyproline and cystathionine.

-   (25) A method for screening a model animal for depression, the    method including the steps of:

collecting a blood sample from a candidate animal, and

measuring a content of one or more compounds selected from the groupconsisting of ADP-RIBOSE, ATP, ADP, AMP, serotonin, tryptophan,kynurenine, SDMA (symmetric dimethylarginine), threonine, glyceric acid,serine, N-acetylaspartic acid, glutamic acid, trigonelline, creatine,2-methyl serine, sphingosine, homovanillic acid, piperidine,sulfoxidated methionine, pipecolic acid, sphinganine, γ-butyrobetaine,guanidoacetic acid, isobutyric acid, creatinine, sarcosine, 3-methylbutyric acid, nicotinamide, betaine, ornithine, carnitine, ethanolamine,phosphoethanolamine, taurine, hypotaurine, aspartic acid, methionine,tyrosine, phosphorylcholine, arginine, asparagine, 3-aminobutyric acid,β-alanine, phenylalanine, lysine, phosphocreatine, alanine, uric acid,choline, isocitric acid, leucine, malic acid, aminoadipic acid,tyramine, valine, glucaric acid, histidine, ADMA (asymmetricdimethylarginine), isoleucine, hydroxyproline and cystathionine in theblood sample.

-   (26) A method for screening a therapeutic agent for patient with    depression, the method including the steps of:

administering a candidate substance for the therapeutic agent to a modelanimal afflicted with the depression,

collecting blood samples before and after the administration of thecandidate substance,

measuring a content of one or more compounds in the blood samples, and

comparing the content of the compound between the blood samples beforeand after the administration of the candidate substance,

wherein the compound is selected from the group consisting ofADP-RIBOSE, ATP, ADP, AMP, serotonin, tryptophan, kynurenine, SDMA(symmetric dimethylarginine), threonine, glyceric acid, serine,N-acetylaspartic acid, glutamic acid, trigonelline, creatine, 2-methylserine, sphingosine, homovanillic acid, piperidine, sulfoxidatedmethionine, pipecolic acid, sphinganine, γ-butyrobetaine, guanidoaceticacid, isobutyric acid, creatinine, sarcosine, 3-methyl butyric acid,nicotinamide, betaine, ornithine, carnitine, ethanolamine,phosphoethanolamine, taurine, hypotaurine, aspartic acid, methionine,tyrosine, phosphorylcholine, arginine, asparagine, 3-aminobutyric acid,β-alanine, phenylalanine, lysine, phosphocreatine, alanine, uric acid,choline, isocitric acid, leucine, malic acid, aminoadipic acid,tyramine, valine, glucaric acid, histidine, ADMA (asymmetricdimethylarginine), isoleucine, hydroxyproline and cystathionine.

-   (27) The method for screening a therapeutic agent for patient with    depression according to Item (26), the method including the steps    of:

administering a candidate substance for the therapeutic agent to a modelanimal afflicted with the depression,

collecting blood samples before and after the administration of thecandidate substance,

measuring a content of one or more compounds in the blood samples, and

comparing the content of the compound between the blood samples beforeand after the administration of the candidate substance,

wherein the compound is selected from the group consisting ofphosphoethanolamine, taurine, hypotaurine, aspartic acid, methionine,tyrosine, phosphorylcholine, arginine, asparagine, 3-aminobutyric acid,β-alanine, phenylalanine, lysine, phosphocreatine, alanine, uric acid,choline, isocitric acid, leucine, malic acid, aminoadipic acid,tyramine, valine, glucaric acid, histidine, ADMA, isoleucine,hydroxyproline and cystathionine.

-   (28) A diagnostic method for diagnosing depression, the method    including the step of measuring a content of one or more compounds    in the blood sample of a subject to be tested,

wherein the compound is selected from the group consisting ofADP-RIBOSE, ATP, ADP, AMP, serotonin, tryptophan, kynurenine, SDMA(symmetric dimethylarginine), threonine, glyceric acid, serine,N-acetylaspartic acid, glutamic acid, trigonelline, creatine, 2-methylserine, sphingosine, homovanillic acid, piperidine, sulfoxidatedmethionine, pipecolic acid, sphinganine, γ-butyrobetaine, guanidoaceticacid, isobutyric acid, creatinine, sarcosine, 3-methyl butyric acid,nicotinamide, betaine, ornithine, carnitine, ethanolamine,phosphoethanolamine, taurine, hypotaurine, aspartic acid, methionine,tyrosine, phosphorylcholine, arginine, asparagine, 3-aminobutyric acid,β-alanine, phenylalanine, lysine, phosphocreatine, alanine, uric acid,choline, isocitric acid, leucine, malic acid, aminoadipic acid,tyramine, valine, glucaric acid, histidine, ADMA (asymmetricdimethylarginine), isoleucine, hydroxyproline and cystathionine.

-   (29) The diagnostic method according to Item (28), the method    including the step of measuring a content of one or more compounds    in the blood sample of the subject,

wherein the compound is selected from the group consisting ofphosphoethanolamine, taurine, hypotaurine, aspartic acid, methionine,tyrosine, phosphorylcholine, arginine, asparagine, 3-aminobutyric acid,β-alanine, phenylalanine, lysine, phosphocreatine, alanine, uric acid,choline, isocitric acid, leucine, malic acid, aminoadipic acid,tyramine, valine, glucaric acid, histidine, ADMA, isoleucine,hydroxyproline and cystathionine.

-   (30) The diagnostic method according to Item (28), the method    including the steps of:

measuring a content of a first compound selected from the groupconsisting of hypotaurine, aspartic acid, methionine, tyrosine,phosphorylcholine, arginine, asparagine, 3-aminobutyric acid, β-alanine,phenylalanine, lysine, phosphocreatine, alanine, uric acid, choline,isocitric acid, leucine, malic acid, aminoadipic acid, tyramine, valine,glucaric acid, histidine, ADMA, isoleucine, hydroxyproline andcystathionine in the blood sample of the subject, and

measuring a content of a second compound selected from the groupconsisting of phosphoethanolamine and taurine in the blood sample of thesubject.

-   (31) The diagnostic method according to Item (28), the method    including the step of measuring a content of aspartic acid and    arginine in the blood sample of the subject.-   (32) The diagnostic method according to Item (28), the method    including the step of measuring a content of aspartic acid and    tyrosine in the blood sample of the subject.-   (33) The diagnostic method according to Item (28), the method    including the step of measuring a content of tyrosine and glucaric    acid in the blood sample of the subject.-   (34) The diagnostic method according to Item (28), the method    including the step of measuring a content of tyrosine and    3-aminobutyric acid in the blood sample of the subject.-   (35) A diagnostic method for diagnosing severity of depression, the    method including the step of measuring a content of cystathionine in    blood sample of a patient.-   (36) The diagnostic method for diagnosing depression according to    Item (28), the method including the step of measuring a content of    one or more compounds in the blood sample of the subject to be    tested,

wherein the compound is selected from the group consisting of ADMA(asymmetric dimethylarginine), hypotaurine, lysine, histidine, leucine,β-alanine, choline, threonine, glyceric acid, isocitric acid, serine,malic acid, 2-methyl serine, sphingosine, homovanillic acid,sulfoxidated methionine, sphinganine, phenylalanine, alanine,aminoadipic acid, N-acetylaspartic acid, glutamic acid, trigonelline,creatine, guanidoacetic acid, isobutyric acid, creatinine, sarcosine,betaine, phosphoethanolamine, taurine, ADP-RIBOSE, aspartic acid,tyrosine, ATP, ADP, asparagine, AMP, serotonin, valine, tryptophan,kynurenine, phosphorylcholine, isoleucine, SDMA (symmetricdimethylarginine), piperidine, pipecolic acid, creatine andnicotinamide.

-   (37) The diagnostic method for diagnosing depression according to    Item (28), the method including the step of measuring a content of    one or more compounds in the blood sample of the subject,

wherein the compound is selected from the group consisting ofphosphoethanolamine, taurine, ADP-RIBOSE, aspartic acid, tyrosine, ATP,ADP, asparagine, AMP, serotonin, valine, tryptophan, kynurenine,phosphorylcholine, isoleucine, SDMA, piperidine, pipecolic acid,creatinine and nicotinamide.

-   (38) The diagnostic method for diagnosing depression according to    Item (28), the method including the step of measuring a content of    one or more compounds in the blood sample of the subject,

wherein the compound is selected from the group consisting of ADMA,hypotaurine, lysine, histidine, leucine, β-alanine, choline, threonine,glyceric acid, isocitric acid, serine, malic acid, 2-methyl serine,sphingosine, homovanillic acid, sulfoxidated methionine, sphinganine andisobutyric acid.

-   (39) The diagnostic method for diagnosing depression according to    Item (28), the method including the step of measuring a content of    one or more compounds in the blood sample of the subject,

wherein the compound is selected from the group consisting ofphenylalanine, alanine, aminoadipic acid, N-acetylaspartic acid,glutamic acid, trigonelline, creatine, guanidoacetic acid, sarcosine andbetaine.

-   (40) A program to cause a computer to execute the steps of:

obtaining a content of a biomarker in a blood sample collected from asubject to be tested,

making a judgement based on the content as to whether the subject iseither one or more of a healthy subject, not a healthy subject, apatient with depression and/or not a patient with depression, and

outputting the result of the judgement,

wherein the biomarker is selected from the group consisting ofADP-RIBOSE, ATP, ADP, AMP, serotonin, tryptophan, kynurenine, SDMA(symmetric dimethylarginine), threonine, glyceric acid, serine,N-acetylaspartic acid, glutamic acid, trigonelline, creatine, 2-methylserine, sphingosine, homovanillic acid, piperidine, sulfoxidatedmethionine, pipecolic acid, sphinganine, γ-butyrobetaine, guanidoaceticacid, isobutyric acid, creatinine, sarcosine, 3-methyl butyric acid,nicotinamide, betaine, ornithine, carnitine, ethanolamine,phosphoethanolamine, taurine, hypotaurine, aspartic acid, methionine,tyrosine, phosphorylcholine, arginine, asparagine, 3-aminobutyric acid,β-alanine, phenylalanine, lysine, phosphocreatine, alanine, uric acid,choline, isocitric acid, leucine, malic acid, aminoadipic acid,tyramine, valine, glucaric acid, histidine, ADMA (asymmetricdimethylarginine), isoleucine, hydroxyproline and cystathionine.

-   (41) The program according to Item (40), further including the step    of causing an apparatus for measuring biomarker to measure the    content of the biomarker in the blood sample.-   (42) A recording medium readable by a computer, the medium storing    the program according to Item (40) or (41).-   (43) The method according to any one of Items (19) to (22), wherein    the depression is atypical depression.-   (44) The method according to Item (23) or (24), wherein the    plurality of patients diagnosed as being afflicted with depression    include a patient diagnosed as being afflicted with atypical    depression.-   (45) The method according to Item (26) or (27), wherein the patient    with depression is a patient afflicted with atypical depression.

DESCRIPTION OF EMBODIMENTS

Hereinafter the embodiments of the present invention accomplished basedon the abovementioned findings are described in detail with reference toExamples. Note that the objective, characteristics, and advantages ofthe present invention as well as the idea thereof will be apparent tothose skilled in the art from the descriptions given herein, and thepresent invention can be easily worked by a person skilled in the artbased on these descriptions. It is to be understood that the embodimentsand specific examples of the invention described hereinbelow are to betaken as preferred examples of the present invention. These embodimentsand examples are presented only for illustrative or explanatorypurposes, and are not intended to restrict the present inventionthereto. It is apparent to those skilled in the art that various changesand modifications may be made based on the descriptions given hereinwithin the intent and scope of the present invention disclosed herein.

==Depression to be Diagnosed Using Biomarker==

As used herein, the “biomarkers for diagnosing depression” (hereinafterthe term “biomarker for diagnosing” is also referred to as “diagnosticmarker”) include the biomarkers that can identify patients withdepression with high probability (disease markers), as well as thebiomarkers that can exclude healthy subjects from the patients withdepression with high probability (health markers).

==Method for Measuring Content of Diagnostic Marker==

The diagnostic marker according to the present invention may bephosphoethanolamine, taurine, hypotaurine, aspartic acid, methionine,tyrosine, phosphorylcholine, arginine, asparagine, 3-aminobutyric acid,β-alanine, phenylalanine, lysine, phosphocreatine, alanine, uric acid,choline, isocitric acid, leucine, malic acid, aminoadipic acid,tyramine, valine, glucaric acid, histidine, ADMA (asymmetricdimethylarginine), isoleucine, hydroxyproline or cystathionine. Whetheror not a patient is afflicted with depression may be diagnosed bymeasuring a content of the diagnostic marker in a blood sample collectedfrom the patient.

When the content of the diagnostic marker in the collected blood sampleis to be measured, the blood sample may be preferably pretreated priorto the measurement. For example, a serum or plasma can be separated fromthe blood by incubation or centrifugation, and the separated serum orplasma may be preferably subjected to the measurement.

The content of a compound as the diagnostic marker in the collectedblood sample may be measured by any known method. Examples of suchmethod include, but not limited to, analysis of the mass of a targetcompound following isolation, such as high-performance liquidchromatography-mass spectrometer (LC-MS) and gas chromatography-massspectrometer (GC-MS). When high-performance liquid chromatography is tobe employed, any of those columns (ex. ion-exchange columns) capable ofsimultaneously analyzing multiple ionic metabolites may be preferablyused.

Other methods for measuring the target compound include, but not limitedto, those of measurement employing NMR analysis, those of measurementemploying acid-base neutralization titration, those of measurementemploying amino acid analyzer, those of measurement with enzymes, thoseof measurement using nucleic acid aptamars/peptide aptamers, those ofmeasurement employing colorimetry, those of measurement only withhigh-performance liquid chromatography, those of measurement only withgas chromatography, and those of measurement only with massspectrometer. When the high-performance liquid chromatography is to beemployed, any of those columns (ex. ion-exchange columns) capable ofsimultaneously analyzing multiple ionic metabolites may be preferablyused.

Use of capillary electrophoresis-time-of-flight mass spectrometer(CE-TOFMS) for the measurement is preferable in that it is capable ofsimultaneously analyzing the in-blood contents of all of the multiplecompounds as the diagnostic markers. When CE-TOFMS is to be employed forthe measurement, the pretreated serum or plasma may be furtherpretreated, preferably as follows.

The serum or plasma is mixed with an alcoholic solvent to terminateenzymatic reactions remaining in the serum or plasma. A preferablealcoholic solvent is methanol. The terminated serum or plasma is mixedwith an organic solvent and water, and the mixture is subjected to phaseseparation and the organic phase containing liposoluble materials suchas phospholipids etc. is removed. The organic solvent to be used may beof any type that can be separated from water by phase separation, andpreferable examples are dichloromethane, chloroform, dichloroethaneetc., and chloroform is particularly preferable. It is preferable thatproteins are removed from the aqueous phase thus obtained. The method toremove proteins may be preferably ultrafiltration but not limitedthereto. After removal of proteins, the alcoholic solvent remaining inthe aqueous phase is preferably removed by distillation. Thedistillation methods for removal of the solvent include, but not limitedto, natural drying, vacuum drying and centrifugal vacuum drying, but thecentrifugal vacuum drying is preferable in that it can be conductedquickly and conveniently. Thus an aqueous solution can be prepared fromthe collected blood sample by removing insoluble materials, and thisaqueous solution from which insoluble materials are removed may bepreferably subjected to a measurement using CE-TOFMS.

The samples to be subjected to measurement with CE-TOFMS may alsoinclude an internal standard to provide criteria for measurements ofelectrophoresis time, content, etc. of compounds as the diagnosticmarker. The internal standard can be any compound that does notinterfere with the efficiency of electrophoresis or mass spectroscopy,and preferable examples include methionine sulfone and 10-camphorsulfonate (10-camphorsulfonic acid, CSA).

The capillary to be used in the capillary electrophoresis is preferablymade of fused-silica. The internal diameter of the capillary ispreferably no more than 100 μm to ensure better separation, and is mostpreferably 50 μm. The length of the capillary is preferably in the rangeof 50 cm to 150 cm.

Fractions containing the compound as the target diagnostic marker amongthose obtained by the capillary electrophoresis may be identified by anymethod, and examples include a method in which the electrophoreticmigration time of the target compound is measured using a sample of thecompound in advance, as well as a method in which relative values of themigration time are compared with that of an internal standard.

As the content of the compound as the target diagnostic marker, the peakarea at the m/z of the target compound in the fraction identified tocontain the compound is then measured. Each peak area can be normalizedby comparing it with the peak area of the internal standard. Theabsolute concentration of the compound as the diagnostic marker in thecollected blood sample can be calculated from the measured peak area bymaking a calibration curve using a sample of the target compound. Thecalibration curve is made preferably by the standard addition methodrather than the standard solution method.

==Use of Diagnostic Marker==

The diagnostic marker can be used in various embodiments of the presentinvention, including those illustrated below.

<Method for Diagnosing Depression>

First, a blood sample is collected from a human or non-human vertebrate.The collected blood sample may be used for diagnosing a disease bymeasuring the content of a diagnostic marker in the blood (hereinafteralso referred to as a marker level). The in-blood content of a biomarkeris preferably the absolute concentration of the biomarker, but may beany value that is correlated with the absolute concentration of thebiomarker and can be used for comparison of the absolute concentrationsamong individuals, and examples include the relative concentration, theweight per unit volume, and the raw data from the measurement ofabsolute concentrations (for example, the value obtained by normalizingthe peak area in a graph from CE-TOFMS measurement).

Prior to the diagnosis, measured are the ranges of the in-bloodbiomarker levels of individuals in a vertebrate species having beendiagnosed as being afflicted with depression (hereinafter referred to asdiseased individuals) and individuals in a vertebrate species havingbeen diagnosed as not having depression (hereinafter referred to ashealthy individuals). If a biomarker level in a blood sample of asubject vertebrate falls within the range of the in-blood biomarkerlevel for the healthy vertebrate, then the probability that thisvertebrate has not been afflicted with the disease is high, whereas ifit falls within the range of the in-blood biomarker level for thediseased vertebrate, then the probability that the animal has beenafflicted with disease is high. The diseased vertebrate may have hadatypical depression. The diseased vertebrate as well as the subjectvertebrate may also be complicated with another disease such as anxietydisorder, borderline personality disorder or panic disorder.

The range of in-blood biomarker level of the healthy vertebrate may beobtained by measuring in advance the range of in-blood biomarker levelsof a subject of a vertebrate species at a time when the same vertebratewas healthy, and this range of biomarker level in the healthy state canbe used when making diagnosis using the marker level at that time of thediagnosis.

The range of the in-blood biomarker level of the healthy vertebrate maybe any range of values appropriate for diagnosing a disease by using adiagnostic marker, and it may be a range between an average plus astandard deviation and the average minus the standard deviation, or arange from the lower limit to the upper limit of the average, in whichthe average can be obtained from multiple measurements on each animal.The most preferable value or range for each diagnostic marker may bechosen for the diagnosis.

Alternatively, a threshold for the in-blood biomarker level may bedetermined to distinguish diseased individuals of a vertebrate speciesfrom healthy individuals of the vertebrate species. For example,in-blood biomarker levels of plurality of diseased individuals andplurality of healthy individuals may be measured to determine thethreshold in advance. The threshold may be determined by any methodknown to those skilled in the art. For example, a threshold can bedetermined so that diseased individuals are included below the threshold(or above the threshold) at a first predetermined ratio and healthyindividuals are included above the threshold (or below the threshold) ata second predetermined ratio. In an exemplary method, a statisticalsoftware such as JMP from SAS Inc. can be used to obtain a thresholdvalue with which chi-square test yields the best result. A diagnosticmarker is preferably provided with high values of both the firstpredetermined ratio and the second predetermined ratio; for example, thevalue may be no less than 70%, more preferably no less than 80%, evenmore preferably no less than 90%, and most preferably 100%. When bothvalues are set high, specificity as well as sensitivity can be high. Ifit is impossible to set both of the first and second predeterminedratios high, the threshold may be determined so that either one of thespecificity and the sensitivity becomes high. The values of both thespecificity and sensitivity are high; for example, the value may be noless than 70%, more preferably no less than 80%, even more preferably noless than 90%, and most preferably 100%. As used herein, the specificityrefers to a percentage value of (the number of healthy individuals aboveor below the threshold)/(the total number of healthy individuals), andthe sensitivity refers to a percentage value of (the number of diseasedindividuals below or above the threshold)/(the total number of diseasedindividuals). Biomarkers having a high specificity can be suitably usedas the biomarker for identifying a patient with depression (hereinafterreferred to as a disease marker) because the probability of a patientbeing afflicted with depression is high if the level of such a biomarkerof the patient is below (or above) the threshold. Biomarker having highsensitivity can be suitably used as the biomarker for excluding ahealthy subject from patients with depression (hereinafter referred toas a health marker) because probability of a patient not being afflictedwith depression is high if the level of such a biomarker is above (orbelow) the threshold.

The diagnostic markers according to the present invention may be used asa disease marker as well as a health marker. Among them,phosphoethanolamine, taurine, aspartic acid, methionine, tyrosine,aminoadipic acid, histidine, isoleucine, ADP-RIBOSE, tyrosine, ATP, ADP,asparagine, AMP, serotonin, valine, tryptophan, kynurenine,phosphorylcholine, SDMA (symmetric dimethylarginine), piperidine,pipecolic acid, creatinine, and nicotinamide are effective as both ofthe disease marker and the health marker, and their plasma concentrationin a subject vertebrate may be measured to make a diagnosis that theprobability of the subject being either afflicted with depression orhealthy is high if the concentration falls in such a certain range asillustrated in Table 1 for human subjects. Among the biomarkers,asparagine, phenylalanine, glucaric acid, hydroxyproline, cystathionine,alanine, aminoadipic acid, N-acetylaspartic acid, glutamic acid,trigonelline, creatine, guanidoacetic acid, sarcosine and betaine aresuitably used as the disease markers, and their plasma concentration ina subject vertebrate may be measured to make a diagnosis that theprobability of the subject being afflicted with depression is high ifthe concentration falls in such a certain range as illustrated in Table1 for human subjects. Further, hypotaurine, phosphorylcholine, arginine,3-aminobutyric acid, β-alanine, lysine, phosphocreatine, alanine, uricacid, choline, isocitric acid, leucine, malic acid, tyramine, valine,ADMA (asymmetric dimethylarginine), threonine, glyceric acid, serine,2-methyl serine, sphingosine, homovanillic acid, sulfoxidatedmethionine, sphinganine, isobutyric acid are suitably used as the healthmarkers, and their plasma concentration in a subject vertebrate may bemeasured to make a diagnosis that the probability of the subject beinghealthy is high if the concentration falls in such a certain range asillustrated for human subjects in either Table 1 derived from Example 1or Table 2 derived from Example 2. When phosphoethanolamine or taurineis to be used as the biomarker, the threshold may be preferably set to2.41 or 50.54 (μM) respectively. The thresholds described herein aremerely examples calculated from a particular population by a particularmethod, and they are likely to vary for different population.

TABLE 1 Threshold Depressive Healthy Biomarker (μM) (Above or below)Phosphoethanolamine 1.99 Below Above Taurine 44.47 Below AboveHypotaurine 2.15 Below Above Aspartic acid 3.95 Below Above Methionine20.33 Below Above Tyrosine 67.19 Below Above Phosphocholine 0.87 BelowAbove Arginine 89.27 Below Above Asparagine 36.26 Below Above3-aminobutyric acid 11.50 Above Below β-alanine 4.47 Below AbovePhenylalanine 43.27 Below Above Lysine 193.52 Below AbovePhosphocreatine 0.93 Above Below Alanine 446.47 Below Above Uric acid332.05 Below Above Choline 28.84 Below Above Isocitric acid 6.90 BelowAbove Leucine 188.26 Below Above Malic acid 2.08 Below Above2-aminoadipic acid 0.70 Below Above Tyramine 0.23 Above Below Valine251.05 Below Above Glucaric acid 0.42 Above Below ADMA 0.33 Below AboveHistidine 76.35 Below Above Isoleucine 30.59 Below Above Hydroxyproline8.81 Below Above Cystathionine 5.63 Above Below

TABLE 2 Threshold Depressive Healthy Biomarker (μM) (Above or below)Phosphoethanolamine 2.06 Below Above Taurine 44.20 Below AboveADP-ribose 0.11 Below Above Aspartic acid 2.88 Below Above Tyrosine66.85 Below Above ATP 1.76 Below Above Methionine 20.12 Below Above ADMA0.33 Below Above Hypotaurine 2.15 Below Above ADP 0.93 Below AboveLysine 193.49 Below Above Phenylalanine 43.27 Below Above Asparagine36.58 Below Above AMP 0.48 Below Above Serotonin 0.11 Below AboveHistidine 90.39 Below Above Valine 223.95 Below Above Leucine 187.94Below Above Alanine 362.35 Below Above Tryptophan 48.66 Below Aboveβ-alanine 3.29 Below Above Aminoadipic acid 0.41 Below Above Kynurenine1.17 Below Above Phosphorylcholine 0.59 Below Above Isoleucine 30.59Below Above SDMA 0.42 Below Above Choline 28.84 Below Above Threonine120.64 Below Above Glyceric acid 16.89 Below Above Isocitric acid 6.90Below Above Serine 128.87 Below Above N-acetylaspartic acid 0.23 BelowAbove Malic acid 2.08 Below Above Glutamic acid 20.05 Below AboveTrigonelline 0.18 Below Above Creatine 0.55 Below Above 2-methyl serine16.65 Below Above Sphingosine 13.38 Below Above Homovanillic acid 2.65Below Above Piperidine 0.04 Below Above Sulfoxidated methionine 3.64Below Above Pipecolic acid 1.10 Below Above Sphinganine 56605.00 BelowAbove γ-butyrobetaine 2.72 Below Above Uric acid 332.05 Below AboveGuanidoacetic add 2.03 Below Above lsobutyric add 2.40 Below AboveCreatinine 60.58 Below Above Sarcosine 2.68 Below Above 3-methyl butyricadd 0.62 Below Above Nicotinamide 0.26 Below Above Betaine 26.13 BelowAbove 3-aminobutyric acid 12.24 Above Below Ornithine 90.34 Below AboveCarnitine 43.03 Below Above Ethanolamine 11.11 Below Above

These biomarkers may be used in combination to improve accuracy ofdiagnosis, and they may be combined in any combination. For example,when two kinds of disease markers are used, if only one of them is at aconcentration indicating the probability of depression, then the subjectmay still be diagnosed as being afflicted with depression at a certainprobability, but if both of the markers are at concentrations indicatingthe probability of depression, then the probability of depression wouldbe higher than that in the case of the one marker. In a preferredembodiment, a health marker is used in combination with a diseasemarker. For example, a subject of an animal individual is diagnosedusing a health marker, and if the health marker is at a concentrationindicating a state of “not being healthy”, then the subject is furtherdiagnosed by using a disease marker. If the disease marker is at aconcentration indicating a state of “being afflicted”, then theprobability of being afflicted with depression is judged to be high,whereas if the disease marker is not at a concentration indicating thestate of “being afflicted”, then the probability of being healthy isjudged to be high. In this way, the accuracy of diagnosis can be madehigher than the case where only disease marker is used. When the healthmarker is at a concentration indicating a state of “being healthy”, thesubject may be diagnosed as being probably healthy, but when anotherdiagnosis is made using a disease marker and if it is at a concentrationindicating a state of “not being afflicted”, then the probability ofbeing healthy would be even higher. If the disease marker is at aconcentration indicating a state of “being afflicted”, then furtherdiagnosis may be made, for example, by analyzing the distance of theconcentration of the biomarkers from their respective thresholds, orjudgement by using a third biomarker.

When identifying subjects with depression using plurality of biomarkers,the health marker may be used as a first biomarker with its thresholdset at a higher level in order to increase the sensitivity, and subjectswith high probability of being healthy may be excluded first. Thethreshold may be set at any high level, for example at about 90%,preferably at about 92%, and even more preferably at about 94%.According to this method, if the health marker at the first stage isproved to be at a concentration indicating a state of being “healthy”,then the subject may be diagnosed as being healthy with highprobability. When the health marker is not at the concentrationindicating the state of being “healthy”, the diagnostic marker may beused in the next step, and if the marker is at a concentrationindicating the state of “being afflicted”, then the probability of beingafflicted with depression is judged to be high, whereas if the marker isnot at the concentration indicating the state of “being afflicted”, thenthe probability of being healthy is judged to be high. In this way, theaccuracy of the judgement can be improved.

The diagnosis using these diagnostic markers may be combined withanother conventional diagnostic method, such as an examination byinterview or questionnaire.

The use of the diagnostic markers as described herein not only enablesdiagnosis with more easiness and higher accuracy at psychiatry, but alsoprovides means for screening at the scenes such as health checkup whereprecise diagnosis cannot be conducted, clinical departments such asinternal medicine and surgery other than psychiatry, and emergencyconditions where verbal communication with a patient is not possible. Inthis way, it becomes possible to bring potential patients withdepression who have been left off the psychiatric examination to thepsychiatry department that should be the first place where they are tobe treated.

By setting the threshold for the sensitivity of a marker at a very highlevel, the marker can be effectively used as a health marker. Thethreshold can be set at any high level, for example at about 90%,preferably at about 92%, and even more preferably at about 94%. At thescene of the health checkup where precise diagnosis cannot be conducted,a diagnosis using such a threshold may be employed as a first diagnosis.In this method, a subject who falls in the range which includes onlysmall number of patients with depression but large number of healthysubjects is diagnosed as being healthy, whereas a patient who falls inthe range which includes large number of patients with depression isdiagnosed as being afflicted with depression at high probability, andthen referred to a psychiatry department for specialized diagnosis. Inthis way, the ratio of patients with depression in the first diagnosiscan be increased, thereby reducing the burden of second diagnosis at thepsychiatry department.

The depression to be diagnosed herein may be of any type that can bediagnosed by a conventional diagnostic method, and preferable examplesare depressions that can be judged according to SCID Interview method.Patients having adjustment disorder are preferably excluded in advanceby a conventional diagnostic method such as an examination by interviewor questionnaire. Patients having anxiety disorder, personalitydisorders such as borderline personality disorder and depressivepersonality disorder or dysthymic disorders can be diagnosed, in whichcase the use of ADMA, hypotaurine, lysine, histidine, leucine,β-alanine, choline, threonine, glyceric acid, isocitric acid, serine,malic acid, 2-methyl serine, sphingosine, homovanillic acid,sulfoxidated methionine, sphinganine, phenylalanine, alanine,aminoadipic acid, N-acetylaspartic acid, glutamic acid, trigonelline,creatine, guanidoacetic acid, sarcosine, betaine, phosphoethanolamine,taurine, ADP-RIBOSE, aspartic acid, tyrosine, ATP, ADP, asparagine, AMP,serotonin, valine, tryptophan, kynurenine, phosphorylcholine,isoleucine, SDMA, piperidine, pipecolic acid, isobutyric acid,creatinine, nicotinamide and the like are preferable, and the thresholdfor them are preferably taken from those shown in Table 2, or thresholdsobtained by the same method as used for Table 2. Patients not havinganxiety disorder, personality disorder such as borderline personalitydisorder and depressive personality disorder, nor dysthymic disorder canbe diagnosed by any of the markers according to the present invention,and the thresholds to be used for them can be taken from both of Table 1and Table 2, but the thresholds shown in Table 1 or thresholds obtainedby the same method as used for Table 1 is more preferable.

<Method for Judging Severity of Depression>

The plasma concentration of cystathionine is inversely proportional tothe scale by CESD as well as to the number of diagnostic match withSCID, and therefore cystathionine is useful as a biomarker for judgingthe severity of depression in a patient with depression.

First, whether or not a subject animal is in depression is judged byusing either any of the biomarkers according to the present invention orany conventional diagnostic method such as the examination by interviewor questionnaire. Then the plasma concentration of the patient withdepression is examined, and if it is, for example, less than 18.36 μM,then a diagnosis of mild depression may be made, whereas if 18.36 μM ormore, then diagnosis of severe depression may be made.

<Judgment of Drug Efficacy>

The effect of a therapeutic agent for certain disease often varies fordifferent individuals. Therefore, knowing the efficacy of thetherapeutic agent for a certain individual is quite beneficial, and itcan be easily achieved by using the diagnostic marker according to thepresent invention. For example, blood samples are collected from adiseased vertebrate before and after administering a therapeutic agentfor depression, and the content of a diagnostic marker in the collectedblood samples is measured to make comparison of the content of thediagnostic marker between the blood samples before and after theadministration of the therapeutic agent. A judgment for the therapeuticagent as being effective can be made if the content of the diagnosticmarker comes closer to the range indicating the state as being healthyafter the administration of the therapeutic agent. In this way, thediagnostic marker according to the present invention can be used toeasily judge whether a therapeutic agent is effective.

<Method for Screening Model Animal for Depression>

By using the diagnostic marker according to the present invention, modelanimals with depression can be screened easily. For example, whether acandidate animal (excluding human) is indeed a model animal fordepression may be judged by a diagnosis of the candidate animal, inwhich the content of the diagnostic marker in the blood of the animal ismeasured and compared with the content of the diagnostic marker in bloodof a healthy animal of the same species.

<Method for Screening Drug with Effectiveness for Depression>

By using experimental animal of depression, a compound that haseffectiveness for treatment of depression can be identified. Forexample, a compound as a candidate of a therapeutic agent for depressionis administered to a model animal (excluding human) of depression, fromwhich blood samples are collected before and after the administration,and the content of a diagnostic marker in the blood samples is measuredto make comparison of the content of the diagnostic marker between theblood samples before and after the administration of the compound. Ajudgment for the administered compound as being effective for treatmentof depression can be made if the content of the diagnostic marker comescloser to the range indicating the state as being healthy after theadministration of the compound. In this way, the diagnostic markeraccording to the present invention can be used to easily screencompounds having effectiveness for treatment of a disease.

<Method for Screening Drug Most Effective for Diseased Animal>

In the case where drugs have different efficacy depending on differentindividuals, a drug that is most effective for a certain individual withdepression can be screened by administering plurality of drugs to theindividual and then conducting diagnosis using the diagnostic marker.

==Computer for Utilizing Biomarker==

After the content of a compound as the diagnostic marker in a bloodsample collected from an individual of a vertebrate species is measuredas described above, the result of the measurement may be transmitted toa computer, and the computer may utilize the results according to any ofthe methods described herein.

For example, a medical practitioner collects a blood sample from anindividual of a vertebrate species and appropriately treats it, thenloads the blood sample on an apparatus for measuring the biomarker. Thecomputer causes an apparatus for measuring the biomarker to measure thecontent of the biomarker in the sample, and obtains the result of themeasurement. Based on the plasma content of the marker thus obtained,the computer may then make a judgement as to whether the tested subjectis either one or more selected from a group consisting of a healthysubject, not a healthy subject, a patient with depression and not apatient with depression. The computer may also make judgements regardingthe severity of a depression, the efficacy of an antidepressant, and thelike, similarly to the method described above. The computer may outputthe result of the judgements thus made, thereby enabling the medicalpractitioner to obtain information about the tested subject.

It should be noted that the program according to the present inventioncauses a computer to execute a method to utilize the plasma content ofthe marker thus obtained. It may also cause a computer to execute thestep of causing the biomarker-measuring apparatus to measure the contentof the biomarker in the sample.

The program may be recorded on a recording medium which is readable by acomputer, and the recording medium may be any of a hard disk, CD, CD,DVD, USB memory, a floppy disk, and the like.

EXAMPLES

Embodiments of the present invention are hereinafter described byreferring to examples, but the scope of the present invention is not tobe limited to the examples described hereinbelow. In this example, it isdemonstrated that patients with depression can be indeed distinguishedfrom healthy subjects by using the marker according to the presentinvention on the patients who have been preliminary diagnosed as beingdepressed.

Example 1 [1] Diagnosis of Depressive Patients

Depressive patients were chosen from the outpatients at Hospital ofNational Center of Neurology and Psychiatry who have been diagnosed asbeing afflicted with depression by SCID (Structured Clinical Interviewfor Diagnosis) Interview Method mainly during their first to thirdexamination, and who have agreed to volunteer under a predeterminedprocedure for informed consent as approved by Ethics Committee. Themajor depression (MD) that is complicated with disease in Axis I or AxisII was excluded. The SCID Interview Method stands for one type ofdifferentiation diagnoses for psychiatric disorders, and is a structuredinterview to enable reproducible diagnosis under the DSM-IV criteria.The first-time diagnosis was conducted to obtain general information byquestioning with regard to lifestyle including smoking habit, drugusage, chief complaint, menstruation and sleeping conditions as well asmedical information such as anamnesis, family history, presence ofphysical disorders and test results for type-B hepatitis, type-Chepatitis, head CT, blood biochemistry, cardiography and radiographs.After informed consents were obtained, questionnaires were made for CESD(a scale for depression) and STAI (a score for anxiety). Missing answerswere checked by conducting a direct interview with a researchcoordinator (a clinical psychologist). In order to obtain the plasmaconcentration of the markers according to the present invention, 14 mLof blood sample was collected from each of the patients, and then plasmawas separated within 2 hours, and stored in liquid nitrogen untilmeasurement. Excluded were the depressive patients being complicatedwith any of the anxiety disorders, patients with mild depression that isdifficult to differentiate from the adjustment disorders, patients withdepression accompanied by borderline personality disorder or anypersonality disorder, and patients having an episode of major depressionduring the development of dysthymic disorder, and the remaining Axis Ipatients and the patients of major depression (MD) having nocomplication with disease in Axis-II were chosen in the total number of35 for the analyses.

Meanwhile, healthy subjects were recruited through advertisements etc.at National Institute of Mental Health and chosen from those who offeredvolunteering. Informed consents were first obtained at a laboratory ofNational Institute of Mental Health, and then the subjects were testedby questioning with regard to lifestyle including smoking habit, drugusage, chief complaint, menstruation, sleeping conditions, anamnesis,family history, presence of physical disorders, CESD and STAI. Those whoscored 21 or more in CESD were excluded as not being healthy subjectsonly by questionnaire. Also, most of the subjects who turned out by theinterview to be under treatment of any physical disorder were excluded.In the questionnaire, missing answers were checked by conducting adirect interview with a research coordinator (a clinical psychologist).The subjects diagnosed as being healthy were thus chosen in the totalnumber of 41. In order to obtain the plasma concentrations of themarkers according to the present invention, 14 mL of blood sample wascollected from each of the subjects, and plasma was separated within 2hours, and stored in liquid nitrogen until measurement.

Table 3 shows sexes, ages and results from the psychiatric tests (CESD,STAI) of the depressive patients as well as the healthy subjects.

TABLE 3 Sex Count Age (SD) CESD (SD) STAI-S (SD) STAI-T (SD) Depressivegroup Male 15 42.1(16.6) 27.7(11.7)  63.1(12.8) 53.5(6.7) Female 2038.0(13.9) 33.7(6.8)  61.8(9.5) 53.5(9.6) Total 35 39.7(14.9) 30.9(9.6)  62.3(10.8) 53.5(8.4) Healthy group Male 17 45.5(11.4) 7.1(3.9)42.1(5.8) 52.0(5.8) Female 24 30.5(12.1) 9.0(5.9) 42.3(7.9) 50.9(8.0)Total 41 36.7(13.9) 8.2(5.2) 42.3(7.0) 51.4(2.8)

With regard to the distribution of the sex, a chi-square test showed nodifference between the groups of depressive patients and healthysubjects. With regard to the age, a t-test showed no significantdifference between the depressive patients and the healthy subjects, orbetween males or between females in each of the groups. No significantdifference was found between both groups with regard to marital status,working status, body height, body weight, weight change in one monthprior to the test, or smoking frequency, either.

In the results of CESD scale determined by a self-report in which thestate of depressive symptom is represented, a t-test showed asignificant difference at p<0.01 between the depressive patients and thehealthy subjects, as well as a significant difference between males orbetween females in each of the groups. No significant difference wasfound between the males and the females in each of the groups. In theresults of STAI-S score that represents the state of anxiety at the timeof interview by a self-report, a t-test showed a significant differenceat p<0.05 between the depressive patients and the healthy subjects, aswell as a significant difference between males and between females ineach of the groups. No significant difference was found between themales and the females in each of the groups. By contrast, in the resultsof STAI-T score that measures the innate likelihood of a subject todevelop anxiety, no significant difference was found between thedepressive patients and the healthy subjects, nor between males orbetween females in each of the groups. No significant difference wasfound between the males and the females in each of the group, either.

Followings are the psychosocial indexes that showed a significantdifference between the two groups. Significantly higher frequency ofinsomnia symptoms of poor falling asleep and early morning awakening wasfound in the depressive group. BMI was slightly larger in the depressivegroup. Frequency for the alcohol intake was slightly higher in thehealthy group. With regard to the level of educational status, while thedepressive group included larger number of junior high school graduates,the healthy group included postgraduates, showing relatively lowereducational status of the depressive group.

Use of medicine at the time of the test yielded a significant difference(p<0.01) in the chi-square test as shown in Table 4 below. The medicinetaken by healthy subjects were painkillers for headache, backache, etc.and antihypertensives, whereas the medicine taken by the depressivepatients was mainly hypnotics and anxiolytics, and SSRI was taken by 7patients.

TABLE 4 Drug usage Not in use In use Total Depressive group 9 26 35Healthy group 30 11 41 Total 38 37 76

[2] Preparation of Plasma Sample

In order to measure the amount in blood of the compound as one of thediagnostic markers by using capillary electrophoresis-time-of-flightmass spectrometer (CE-TOFMS), the plasma samples for CE-TOFMSmeasurement were prepared in the method described below.

Blood samples were collected from the patients under informed consent(conducted at National Center of Neurology and Psychiatry), and 100 μLeach of plasma was prepared and put into a centrifuge tube. As aninternal standard solution, 0.45 mL of methanol (Wako Pure Chemical,LC/MS grade) containing 10 μM of methionine sulfone and 10 μM of10-camphor sulfonate (H3304-1002, purchased from Human MetabolomeTechnologies, Inc.) was added. After addition of 0.5 mL of chloroform(Wako Pure Chemical, reagent grade) and 200 μL of Milli-Q water, thesample was vigorously mixed by vortex for 30 sec., and centrifuged (4°C., 2300×g, 5 min). The aqueous phase was transferred to aultrafiltration unit (Millipore, Ultrafree-MCPBCC centrifugal filterunit, 5 kDa) and filtered by centrifugation (4° C., 9100×g, 2 to 4hours) until almost no solution was remained in the filter cup. Afterthe filter cup was removed, the filtrate was dried by centrifugationunder vacuum. The dried material was redissolved in 50 μL of Milli-Qwater containing the internal standard to prepare the sample for themeasurement by CE-TOFMS.

[3] Method for CE-TOFMS Measurement and Analysis of Result

The CE-TOFMS measurement and the following data analyses were conductedon Agilent CE-TOF-MSD System (Agilent Technologies Inc.) using fusedsilica capillaries.

The conditions for the CE-TOFMS measurement were as follows:

(A) Cation Mode

Run buffer: Cation Buffer Solution (p/n: H3301-1001)

Rinse buffer: Cation Buffer Solution (p/n: H3301-1001)

Sample injection: Pressure injection 50 mbar, 10 sec

CE voltage: Positive, 30 kV

MS ionization: ESI Positive

MS capillary voltage: 4,000 V

MS scan range: m/z 50-1,000

Sheath liquid: HMT Sheath Liquid (p/n: H3301-1020)

(B) Anion Mode

Run buffer: Anion Buffer Solution (p/n: H3302-1021)

Rinse buffer: Anion Buffer Solution (p/n: H3302-1022)

Sample injection: Pressure injection 50 mbar, 25 sec

CE voltage: Positive, 30 kV

MS ionization: ESI Negative

MS capillary voltage: 3,500 V

MS scan range: m/z 50-1,000

Sheath liquid: HMT Sheath Liquid (p/n: H3301-1020)

From the peaks detected by CE-TOFMS, m/z values, migration times (MT)and area values were obtained as the peak information. The peak areavalues obtained were converted to relative area values by Formula 1below.

$\begin{matrix}{{{Relative}\mspace{14mu}{area}} = \frac{{Area}\mspace{14mu}{of}\mspace{14mu}{target}\mspace{14mu}{peak}}{{Area}\mspace{14mu}{of}{\mspace{11mu}\;}{internal}\mspace{14mu}{standard}}} & \left\lbrack {{Formula}\mspace{14mu} I} \right\rbrack\end{matrix}$

[4] Measurement of Plasma Concentration of Diagnostic Marker by CE-TOFMSand Analysis of Result

Blood samples were collected from the depressive patients and thehealthy subjects, and the samples for the CE-TOFMS measurement wereprepared from the plasma, and subjected to the measurement by CE-TOFMSunder cation mode and anion mode. From these measurements, peak areaswere obtained as the data representing the content of each compound ineach of the samples.

From the data of peak areas, a value that yields the best chi-squarevalue of judgement was chosen using the statistical software JMP fromSAS Inc. as the threshold of the disease marker to distinguishdepressive patients from healthy subjects. Also, using commerciallyavailable samples, a calibration curve was made and used to convert thethresholds thus obtained to absolute values (threshold (μM)) based onthe concentration in the plasma. Table 5 shows the thresholds for thedisease markers used and the numbers of the healthy subjects anddepressive patients fall in the range above or below the thresholds, andthe results of the calculation of specificity and sensitivity based onthese values are shown in Table 6.

TABLE 5 Above Below Phosphoethanolamine Depressive 3 32 Healthy 33 8Taurine Depressive 8 27 Healthy 35 6 Hypotaurine Depressive 2 33 Healthy19 22 Aspartic acid Depressive 1 34 Healthy 26 15 Methionine Depressive6 29 Healthy 28 13 Tyrosine Depressive 1 34 Healthy 25 16 PhosphocholineDepressive 0 35 Healthy 12 29 Arginine Depressive 1 34 Healthy 17 24Aspartic acid Depressive 16 19 Healthy 34 7 3-aminobutyric acidDepressive 33 2 Healthy 24 17 β-alanine Depressive 1 34 Healthy 13 28Phenylalanine Depressive 21 14 Healthy 39 2 Lysine Depressive 3 32Healthy 21 20 Phosphocreatine Depressive 35 0 Healthy 32 9 AlanineDepressive 1 34 Healthy 13 28 Uric acid Depressive 0 35 Healthy 12 29Choline Depressive 3 32 Healthy 17 24 Isocitric acid Depressive 1 34Healthy 13 28 Leucine Depressive 1 34 Healthy 16 25 Malic acidDepressive 1 34 Healthy 13 28 Aminoadipic acid Depressive 7 28 Healthy25 16 Tyramine Depressive 35 0 Healthy 36 5 Valine Depressive 5 30Healthy 22 19 Glucaric acid Depressive 6 29 Healthy 0 41 ADMA Depressive0 35 Healthy 12 29 Histidine Depressive 11 24 Healthy 31 10 IsoleucineDepressive 7 28 Healthy 25 16 Hydroxyproline Depressive 17 18 Healthy 2813 Cystathionine Depressive 34 1 Healthy 34 7

TABLE 6 Performance when used a sole marker Threshold Depressive HealthySpecificity Sensitivity Threshold (μM) (Above or below)Phosphoethanolamine 80.5% 91.4% 0.0059 1.99 Below Above Taurine 85.4%77.1% 0.1133 44.47 Below Above Hypotaurine 46.3% 94.3% 0.0113 2.15 BelowAbove Aspartic acid 63.4% 97.1% 0.0316 3.95 Below Above Methionine 68.3%82.9% 0.2743 20.33 Below Above Tyrosine 61.0% 97.1% 0.8549 67.19 BelowAbove Phosphocholine 29.3% 100.0% 0.0099 0.87 Below Above Arginine 41.5%97.1% 1.6627 89.27 Below Above Asparagine 82.9% 54.3% 0.4149 36.26 BelowAbove 3-aminobutyric acid 41.5% 94.3% 0.0038 11.50 Above Below β-alanine31.7% 97.1% 0.0398 4.47 Below Above Phenylalanine 95.1% 40.0% 1.003843.27 Below Above Lysine 51.2% 91.4% 2.5304 193.52 Below AbovePhosphocreatine 22.0% 100.0% 0.0022 0.93 Above Below Alanine 31.7% 97.1%4.6678 446.47 Below Above Uric acid 29.3% 100.0% 1.1712 332.05 BelowAbove Choline 41.5% 91.4% 0.5004 28.84 Below Above Isocitric acid 31.7%97.1% 0.0462 6.90 Below Above Leucine 39.0% 97.1% 3.7470 188.26 BelowAbove Malic acid 31.7% 97.1% 0.0456 2.08 Below Above 2-aminoadipic acid61.0% 80.0% 0.0097 0.70 Below Above Tyramine 12.2% 100.0% 0.0034 0.23Above Below Valine 53.7% 85.7% 4.5583 251.05 Below Above Glucaric acid100.0% 17.1% 0.0021 0.42 Above Below ADMA 29.3% 100.0% 0.00113 0.33Below Above Histidine 75.6% 68.6% 1.2351 76.35 Below Above Isoleucine61.0% 80.0% 1.7544 30.59 Below Above Hydroxyproline 68.3% 51.4% 0.08498.81 Below Above Cystathionine 97.1% 17.1% 0.0008 5.63 Above Below

Among the compounds listed, hypotaurine, phosphorylcholine, arginine,3-aminobutyric acid, β-alanine, lysine, phosphocreatine, alanine, uricacid, choline, isocitric acid, leucine, malic acid, tyramine, valine,ADMA (asymmetric dimethylarginine) etc. have particularly highsensitivity, indicating their potential for excluding healthy subjects,and therefore they can be preferably used as health markers. Meanwhile,asparagine, phenylalanine, glucaric acid, hydroxyproline, cystathionineetc. have particularly high specificity, indicating their potential forfinding patients, and therefore they can be preferably used as diseasemarkers. In particular, the compounds having both the sensitivity andspecificity over 60%, i.e. phosphoethanolamine, taurine, aspartic acid,methionine, tyrosine, aminoadipic acid, histidine, isoleucine etc. areuseful as both the disease marker and the health marker, and each one ofthem may be used solely for diagnosis.

Since the contents of these compounds in blood can be easily measuredall at once by simply analyzing a blood sample by CE-TOFMS, the resultsfrom plurality of these compounds can be quite easily combined to makediagnosis.

[5] Use of Health Marker for Effectively Excluding Healthy Subject

In this example, the abovementioned compounds were used as biomarkerswherein the threshold for their sensitivity as health markers was set to94.3%. The numbers of depressive patients or healthy subjects includedin the ranges above the threshold or below the threshold are shown inTable 7, and the results of the calculation for their specificity areshown in Table 8.

TABLE 7 Marker Above Below Aspartic acid Depressive 2 33 Healthy 28 13Methionine Depressive 2 33 Healthy 19 22 Tyrosine Depressive 2 33Healthy 26 15 Phosphocholine Depressive 2 33 Healthy 16 25 ArginineDepressive 2 33 Healthy 17 24 Asparagine Depressive 2 33 Healthy 13 283-aminobutyric acid Depressive 33 2 Healthy 24 17 β-alanine Depressive 233 Healthy 13 28 Phenylalanine Depressive 2 33 Healthy 15 26 LysineDepressive 2 33 Healthy 13 28 Phosphocreatine Depressive 33 2 Healthy 2615 Alanine Depressive 2 33 Healthy 13 28 Uric acid Depressive 2 33Healthy 13 28 Choline Depressive 2 33 Healthy 13 28 Isocitric acidDepressive 2 33 Healthy 13 28 Leucine Depressive 2 33 Healthy 17 24Malic acid Depressive 2 33 Healthy 14 27 2-aminoadipic acid Depressive 233 Healthy 8 33 Tyramine Depressive 33 2 Healthy 33 8 Valine Depressive2 33 Healthy 12 28 Glucaric acid Depressive 33 2 Healthy 33 8 ADMADepressive 2 33 Healthy 15 26 Histidine Depressive 2 33 Healthy 16 25Isoleucine Depressive 2 33 Healthy 16 25

TABLE 8 Specificity when sensitivity is set to 94.3% ThresholdSpecificity Sensitivity Threshold (um) Aspartic acid 68.3% 94.3% 0.03063.832 Methionine 46.3% 94.3% 0.2986 22.134 Tyrosine 63.4% 94.3% 0.850666.854 Phosphocholine 39.0% 94.3% 0.0087 0.761 Arginine 41.5% 94.3%1.6627 89.272 Asparagine 31.7% 94.3% 0.5623 49.144 3-aminobutyric acid41.5% 94.3% 0.0038 11.625 β-alanine 31.7% 94.3% 0.0398 4.469Phenylalanine 36.6% 94.3% 1.4551 62.729 Lysine 31.7% 94.3% 2.8313216.536 Phosphocreatine 36.6% 94.3% 0.0026 1.105 Alanine 31.7% 94.3%4.6678 446.472 Uric acid 31.7% 94.3% 1.1507 326.248 Choline 31.7% 94.3%0.5303 30.565 Isocitric acid 31.7% 94.3% 0.0462 6.899 Leucine 41.5%94.3% 3.5795 179.846 Malic acid 34.1% 94.3% 0.0431 1.965 Aminoadipicacid 19.5% 94.3% 0.0144 0.702 Tyramine 19.5% 94.3% 0.0036 0.242 Valine29.3% 94.3% 5.1873 285.691 Glucaric acid 19.5% 94.3% 0.0006 0.128 ADMA39.0% 94.3% 0.0106 0.305 Histidine 36.6% 94.3% 1.4057 86.896 Isoleucine39.0% 94.3% 2.1169 36.914

Determination of a threshold in this way makes it possible to secludedepressive patients to a range either above or below the threshold.Thus, the ratio of depressive patients among total subjects can beincreased by adopting a range that contains a larger number ofdepressive patients.

Practically, in the case where the sensitivity is set to 94.3%, as longas the specificity is 5.7 (=100−94.3)% or higher, this method canincrease the ratio of depressive patients in a population regardless ofthe ratios of depressive patients and healthy subjects. All of thebiomarkers listed in Table 8 have specificity of 5.7% or higher, andtherefore are useful as the health markers.

[6] Diagnosis Using Plurality of Biomarkers

The accuracy of diagnosis can be improved by using plurality ofbiomarkers among the abovementioned compounds. Effective combinationsare considered to be, in particular, the combination of a disease markerand a health marker, the combination of a biomarker effective for bothand a disease marker, the combination of a biomarker effective for bothand a health marker, and the combination of two biomarkers effective forboth. Their specific examples are as follows:

-   (1) Where the patients with aspartic acid less than 3.95 μM and    arginine less than 89.27 μM are defined as depressive and the others    are defined as healthy, the sensitivity is 94% and the specificity    is 88%.-   (2) Where the patients with aspartic acid less than 3.95 μM and    tyrosine less than 67.19 μM are defined as depressive and the others    are defined as healthy, the sensitivity is 94% and the specificity    is 81%.-   (3) Where the patients with tyrosine less than 67.19 μM and glucaric    acid 0.42 μM or more are defined as depressive and the others are    defined as healthy, the sensitivity is 94% and the specificity is    81%.-   (4) Where the patients with tyrosine less than 67.19 μM and    3-aminobutyric acid less than 11.50 μM are defined as depressive and    the others are defined as healthy, the sensitivity is 91% and the    specificity is 85%.    Thus, the accuracy of diagnosis is improved in comparison to the    case where each of the biomarkers is used alone.

[7] Biomarker for Judging Severity of Depression

Similar to Example 4, the content of cystathionine in each sample fromthe depressive patient group was obtained in the form of peak area data,and was tested by Spearman's rank correlation coefficient in relation tothe CESD scale as well as the number of diagnostic matches in SCID.Inverse correlation was found with CESD by r=−0.460 and p=0.039, andwith SCID by r=−0.339 and p=0.049.

Then, the threshold of the cystathionine content between mildlydepressive patients and severely depressive patients was analyzed, andit was found that the mildly depressive patients and severely depressivepatients could be distinguished at the threshold of 0.00265 as shown inTable 9. In this example, those who scored 30 or less in CESD weredefined as being afflicted with mild depression, whereas those of 31 orhigher were defined as severe depression, and those who matched 5 to 6items out of the 9 criteria in SCID were defined as mild depression,whereas those of 7 or more matches were defined as severe depression.

TABLE 9 Above Below CESD-mild 4 14 CESD-severe 13 4 SCID-mild 5 13SCID-severe 8 9

Thus, cystathionine can be effectively used as a diagnostic marker fordepression, in particular, the biomarker for judging the severity ofdepression.

It should be noted that the threshold could be converted to an absoluteconcentration of about 18.36 μM.

Example 2 [1] Diagnosis of Depressive Patients

In this example, the analyses were conducted on the patient groupconsisting of 34 Axis I patients including 3 depressive patientscomplicated with anxiety disorders other than adjustment disorder and 2depressive patients complicated with borderline personality disorder, aswell as the control group including 7 patients with mild depressionwhich was difficult to distinguish from adjustment disorder, and 31healthy subjects. The subjects in the patient group and the controlgroup were chosen in the similar way to Example 1. Between both groups,no significant difference was found in terms of age, body height, bodyweight, BMI and sex. There was no significant difference in maritalstatus, working status, weight change in one month prior to the test, orsmoking frequency, either.

[2] Methods for Preparation of Plasma Sample, Measurement by CE-TOFMSand Analysis of Result

The methods for preparation of plasma samples from the abovementionedsubjects to be tested and the measurement of each marker in the samplesby CE-TOFMS were the same as those in Example 1.

[3] Measurement of Plasma Concentration of Diagnostic Marker by CE-TOFMSand Analysis of Result

The measurement of the concentration of diagnostic markers in the plasmaof the subjects from the patient group and the control group,determination of the thresholds, and the analyses of the results wereconducted in the similar way to Example 1.

Table 10 shows the results of the analyses for each of the diagnosticmarkers.

TABLE 10 Below threshold Above threshold Threshold Depression HealthyDepression Healthy Biomarker Threshold (concentration) patient subjectpatient subject Sensitivity Specificity Phosphoethanolamine 0.0061 2.0628 2 6 36 82.4% 94.7% Taurine 0.1126 44.20 26 4 8 34 76.5% 89.5%ADP-ribose 0.0005 0.11 32 11 2 27 94.1% 71.1% Aspartic acid 0.023 2.8821 3 13 35 61.8% 92.1% Tyrosine 0.8506 66.85 33 13 1 25 97.1% 65.8% ATP0.0063 1.76 28 6 6 32 82.4% 84.2% Methionine 0.2714 20.12 29 13 5 2585.3% 65.8% ADMA 0.0113 0.33 34 24 0 14 100.0% 36.8% Hypotaurine 0.01132.15 33 16 1 22 97.1% 57.9% ADP 0.0049 0.93 28 8 6 30 82.4% 78.9% Lysine2.53 193.49 30 16 4 22 88.2% 57.9% Phenylalanine 1.0038 43.27 14 1 20 3741.2% 97.4% Asparagine 0.4186 36.58 21 5 13 33 61.8% 86.8% AMP 0.00190.48 30 14 4 24 88.2% 63.2% Serotonin 0.0016 0.11 22 5 12 33 64.7% 86.8%Histidine 1.4623 90.39 34 27 0 11 100.0% 28.9% Valine 4.0663 223.95 21 613 32 61.8% 84.2% Leucine 3.7406 187.94 32 21 2 17 94.1% 44.7% Alanine3.7883 362.35 19 6 15 32 55.9% 84.2% Tryptophan 0.9408 48.66 26 11 8 2776.5% 71.1% β-alanine 0.0293 3.29 30 17 4 21 88.2% 55.3% Aminoadipicacid 0.0057 0.41 9 0 25 38 26.5% 100.0% Kynurenine 0.0256 1.17 31 15 323 91.2% 60.5% Phosphorylcholine 0.0067 0.59 24 11 10 27 70.6% 71.1%Isoleucine 1.7544 30.59 27 14 7 24 79.4% 63.2% SDMA 0.01 0.42 25 12 9 2673.5% 68.4% Choline 0.5004 28.84 31 21 3 17 91.2% 44.7% Threonine 1.9038120.64 34 28 0 10 100.0% 26.3% Glyceric acid 0.0473 16.89 31 19 3 1991.2% 50.0% Isocitric acid 0.0462 6.90 32 22 2 16 94.1% 42.1% Serine1.0526 128.87 29 17 5 21 85.3% 55.3% N-acetylaspartic acid 0.0012 0.2312 2 22 36 35.3% 94.7% Malic acid 0.0456 2.08 32 22 2 16 94.1% 42.1%Glutamic acid 0.2428 20.05 13 3 21 35 38.2% 92.1% Trigonelline 0.00340.18 12 1 22 37 35.3% 97.4% Creatine 0.193 0.55 7 0 27 38 20.6% 100.0%2-methyl serine 0.2012 16.65 29 21 5 17 85.3% 44.7% Sphingosine 13.382513.38 28 16 6 22 82.4% 57.9% Homovanillic acid 0.0109 2.65 34 32 0 6100.0% 15.8% Piperidine 0.0006 0.04 23 10 11 28 67.6% 73.7% Suffoxidatedmethionine 0.0216 3.64 34 30 0 8 100.0% 21.1% Pipecolic acid 0.0263 1.1022 13 12 25 64.7% 65.8% Sphinganine 56605 56605.00 28 16 6 22 82.4%57.9% γ-butyrobetaine 0.0671 2.72 34 30 0 8 100.0% 21.1% Uric acid1.1712 332.05 34 26 0 12 100.0% 31.6% Guanidoacetic acid 0.0264 2.03 144 20 34 41.2% 89.5% Isobutyric acid 0.0517 2.40 28 20 6 18 82.4% 47.4%Creatinine 0.9358 60.58 24 15 10 23 70.6% 60.5% Sarcosine 0.0304 2.68 166 18 32 47.1% 84.2% 3-methyl butyric acid 0.0472 0.62 29 20 5 18 85.3%47.4% Nicotinamide 0.0024 0.26 25 15 9 23 73.5% 60.5% Betaine 0.611226.13 10 2 24 36 29.4% 94.7% 3-aminobutyric acid 0.004 12.24 34 30 0 8100.0% 21.1% Ornithine 1.5677 90.34 32 26 2 12 94.1% 31.6% Carnitine1.3303 43.03 26 18 8 20 76.5% 52.6% Ethanolamine 0.0492 11.11 31 26 3 1291.2% 31.6%

Among the compounds listed, ADMA, hypotaurine, lysine, histidine,leucine, β-alanine, choline, threonine, glyceric acid, isocitric acid,serine, malic acid, 2-methyl serine, sphingosine, homovanillic acid,sulfoxidated methionine, sphinganine, isobutyric acid etc. haveparticularly high sensitivity, indicating their potential for excludinghealthy subjects, and therefore they can be preferably used as healthmarkers. Meanwhile, phenylalanine, alanine, aminoadipic acid,N-acetylaspartic acid, glutamic acid, trigonelline, creatine,guanidoacetic acid, sarcosine, betaine etc. have particularly highspecificity, indicating their potential for identifying patients, andtherefore they can be preferably used as disease markers. In particular,the compounds having both the sensitivity and specificity over 60%, i.e.phosphoethanolamine, taurine, ADP-RIBOSE, aspartic acid, tyrosine, ATP,ADP, asparagine, AMP, serotonin, valine, tryptophan, kynurenine,phosphorylcholine, isoleucine, SDMA (symmetric dimethylarginine),piperidine, pipecolic acid, creatinine, nicotinamide etc. are useful asboth the disease marker and the health marker, and each one of them maybe used solely for diagnosis.

[4] Set of Health Marker and its Threshold for Effectively ExcludingHealthy Subject

This example demonstrates that health markers can effectively excludehealthy subjects.

When the threshold for each of the biomarkers was set so that theirsensitivity becomes 94.1%, an exclusion of the healthy subjects waseffectively achieved by excluding a population containing smaller numberof depressive patients. The specificity was calculated at the threshold,and the results are shown in Table 11.

TABLE 11 Below threshold Above threshold Threshold Depression HealthyDepression Healthy Biomarker Threshold (concentration) patient subjectpatient subject Sensitivity Specificity ADP-Ribose 0.0005 0.11 2 32 2315 94.1% 74.2% Tyrosine 0.8506 66.85 2 32 22 16 94.1% 71.0% Hypotaurine0.0113 2.14 2 32 17 21 94.1% 54.8% Methionine 0.3008 22.30 2 32 16 2294.1% 51.6% ADMA 0.0103 0.30 2 32 16 22 94.1% 51.6% β-Alanine 0.03343.75 2 32 15 23 94.1% 48.4% Aspartic acid 0.0354 4.42 2 32 15 23 94.1%48.4% Asparagine 0.5330 46.58 2 32 14 24 94.1% 45.2% Taurine 0.170566.91 2 32 14 24 94.1% 45.2% Leucine 3.7470 188.26 2 32 14 24 94.1%45.2% Uric acid 1.1407 323.41 2 32 14 24 94.1% 45.2% Serotonin 0.00500.34 2 32 13 25 94.1% 41.9% Malic acid 0.0456 2.08 2 32 13 25 94.1%41.9% Isocitric acid 0.0462 6.90 2 32 13 25 94.1% 41.9% Lysine 2.8313216.53 2 32 12 26 94.1% 38.7% Histidine 1.4057 86.90 2 32 12 26 94.1%38.7% Choline 0.5303 30.56 2 32 12 26 94.1% 38.7% Phenylalanine 1.455162.72 2 32 12 26 94.1% 38.7% Glutamic acid 0.5787 47.79 2 32 12 26 94.1%38.7% Succinic acid 0.0390 8.36 2 32 12 26 94.1% 38.7% Alanine 4.6678446.47 2 32 11 27 94.1% 35.5% Phosphoethanolamine 0.0106 3.58 2 32 11 2794.1% 35.5% Phosphorylcholine 0.0099 0.87 2 32 11 27 94.1% 35.5% Serine1.1198 137.10 2 32 11 27 94.1% 35.5% Arginine 1.6627 89.27 2 32 11 2794.1% 35.5% Valine 5.3006 291.93 2 32 10 28 94.1% 32.3% γ-Butyrobetaine0.0652 2.64 2 32 10 28 94.1% 32.3% Isoleucine 2.2144 38.61 2 32 10 2894.1% 32.3% N-Acetylaspartic acid 0.0019 0.37 2 32 10 28 94.1% 32.3%Lactic acid 13.2742 4297.40 32 2 10 28 94.1% 32.3% Ornithine 1.567790.34 2 32 10 28 94.1% 32.3% Sphinganine 1p 281.4486 281.45 2 32 10 2894.1% 32.3% Sphingosine 1803.3079 1803.31 2 32 10 28 94.1% 32.3%Sphingosine 1p 1803.3079 1803.31 2 32 10 28 94.1% 32.3% Aminoadipic acid0.0142 1.02 2 32 9 29 94.1% 29.0% Phosphocreatine 0.0026 1.11 32 2 8 3094.1% 25.8% Tyramine 0.0037 0.25 32 2 7 31 94.1% 22.6%

Determination of a threshold in this way makes it possible to secludedepressive patients to a range either above or below the threshold.Thus, the ratio of depressive patients among total subjects can beincreased by adopting the range which contains a larger number ofdepressive patients.

Practically, in the case where the sensitivity is set to 94.1%, as longas the specificity is 5.9 (=100−94.1)% or higher, this method canincrease the ratio of depressive patients in a population regardless ofthe ratios of depressive patients and healthy subjects. All of thebiomarkers listed in Table 8 have specificity of 5.7% or higher, andtherefore are useful as the health markers. All of the biomarkers listedin Table 11 have specificity of 5.9% or higher, and therefore are usefulas the health markers.

Example 3 [1] Calculation of Plasma Marker Concentration Using StandardAddition Method

In Examples 1 and 2, the calibration curve was made from thecommercially available samples as the standard solutions and used toconvert the obtained thresholds to absolute values (threshold (μM))based on the plasma concentration (standard solution method). In thisexample, standard solutions were prepared at a predetermined series ofconcentrations, and for each of the samples, a series of the solutionsadded with each of the standard solutions were prepared to make acalibration curve, and then the plasma marker concentration of each ofthe samples in Example 2 was determined (standard addition method).Using the concentrations thus obtained, thresholds were determined inthe similar way to Example 2, and the resulting threshold forphosphoethanolamine and taurine was 2.41 and 50.54 (μM), respectively.

[2] Change in Plasma Marker Concentration Before and After Treatment

Plasma marker concentrations (μM) of phosphoethanolamine and taurinewere measured in 2 depressive patients, who were not included in thepopulations in Examples 1 and 2, before treatment (pre-treatment) andafter a diagnosis as remission of the depression by treatment usingmedicine such as SSRI for about 6 months (post-treatment). Theconcentrations were determined by the standard addition method.

TABLE 12 Phosphoethanolamine Taurine Before After Before After treatmenttreatment treatment treatment Patient “A” 2.0 3.5 39.0 55.4 Patient “B”2.2 3.8 44.6 100.1

Thus, the plasma marker concentrations of phosphoethanolamine andtaurine in both patients increased to a level comparable to that of thehealthy subjects after the treatment. By using the thresholds determinedin [1] for example, both the Patient A and Patient B can be diagnosed asbeing afflicted with depression before the treatment, and as beinghealthy after the treatment, even using either of the markers.

[3] Validation of Threshold I

Plasma marker concentrations in 11 individuals independent of thosetested in Examples 1 and 2 (of whom 6 have been definitively diagnosedas being healthy subjects and 5 as patients with depression) weremeasured in a similar way to [1] above, and diagnosis was made using thethresholds determined in [1].

TABLE 13 Marker diagnosis Subject Definitive diagnosisPhosphoethanolamine Taurine 1 Depression Depressive Depressive 2Depression Depressive Depressive 3 Depression Depressive Depressive 4Depression Depressive Healthy 5 Depression Healthy Healthy 6 HealthyHealthy Healthy 7 Healthy Healthy Healthy 8 Healthy Healthy Healthy 9Healthy Healthy Healthy 10 Healthy Healthy Healthy 11 Healthy HealthyHealthy

Using the thresholds determined in [1], an appropriate diagnosis couldbe made for the healthy subjects with a probability of 100%. Anappropriate diagnosis could be made for the patients with depressionwith a probability of 80% when using phosphoethanolamine, and with aprobability of 60% when using taurine. Therefore, phosphoethanolamineand taurine are useful as the diagnostic markers of depression.

[4] Validation of Threshold II

Plasma marker concentrations in 14 individuals independent of thosetested in Examples 1 to 3 (All have panic disorder. Of them, 9individuals have been definitively diagnosed as not being patients withdepression [marked as healthy subjects in the table], and 5 as beingpatients of atypical depression) were measured in a similar way to [1]above, and diagnosis was made using the thresholds determined in [1].

TABLE 14 Marker diagnosis Subject Definitive diagnosisPhosphoethanolamine Taurine 1 Atypical depression Depressive Depressive2 Atypical depression Depressive Depressive 3 Atypical depressionDepressive Depressive 4 Atypical depression Depressive Healthy 5Atypical depression Depressive Healthy 6 Healthy Healthy Depressive 7Healthy Healthy Healthy 8 Healthy Healthy Healthy 9 Healthy HealthyHealthy 10 Healthy Healthy Healthy 11 Healthy Healthy Healthy 12 HealthyHealthy Healthy 13 Healthy Healthy Healthy 14 Healthy Healthy Healthy

Using the thresholds determined in [1], an appropriate diagnosis couldbe made for both the depressive patients and non-depressive patientswith a probability of 100% when using phosphoethanolamine. In the caseof taurine, an appropriate diagnosis could be made for depressivepatients with a probability of 60% and non-depressive patients with aprobability of 89%. Therefore, phosphoethanolamine and taurine areuseful as diagnostic markers of depression for the patients withatypical depression as well. Further, the markers according to thepresent invention can be used even if depression is complicated withother disease (for example, panic disorder).

INDUSTRIAL APPLICABILITY

The present invention can provide the biomarkers for diagnosingdepression and the diagnostic methods using the biomarkers.

What is claimed:
 1. A method for treating depression using a biomarker,comprising the steps of: (i) measuring a level of phosphoethanolamine ina blood sample collected from a subject; (ii) comparing the level ofphosphoethanolamine in the blood sample with a predetermined threshold;(iii) in response to the level of phosphoethanolamine in the bloodsample being below the predetermined threshold, determining by aphysician or a psychiatrist that the subject requires a treatment fordepression; and (iv) treating the subject determined to suffer fromdepression in step (iii).
 2. The method according to claim 1, whereinthe predetermined threshold is no greater than 2.41 μM.
 3. The methodaccording to claim 1, wherein the treatment in the step (iv) comprisesthe use of a medicine.
 4. The method according to claim 3, wherein themedicine comprises Selective Serotonin Reuptake Inhibitors (SSRI).